The Neutrals Quality Control Reference Material (QCRM), the Revered-Phase

QCRM, LCMS QCRM, and HILIC QCRM are part of the Reference Material Portfolio.

They are qualitative tools that allow the user to better evaluate and benchmark

their HPLC, UPLC®, or LC/MS system before analysis of critical material. These

particular standards are specially formulated depending on the customer need and

instrumentation being utilized. The compounds were vigorously evaluated and chosen

because they provide the following advantages:

■■ Assess system readiness on a regular basis

■■ Characterize the benchmark performance easily and reproducibly

■■ Range from simple mixtures to more complex

■■ Acceptable for use on a variety of columns and systems under different conditions

It is highly recommended that every customer who is running an HPLC, UPLC, or LC/MS

system regardless of the type of lab, use one of these standards as an initial benchmark/

performance test as well as on a regular basis to continually monitor the health of their

system to add confidence in real sample results.

Quality Control Reference Material Products

2

Figure 1. Example of the chromatography obtained for the Neutrals QCRM mixture via UV using a BEH C18, 1.7 μm, 2.1 x 50 mm.

10 mM Ammonium Formate pH 3; Acetonitrile(50/50)

20 mM Potassium Phosphate pH 7; Acetonitrile(50/50)

10 mM Ammonium Bicarbonate pH 10; Acetonitrile(50/50)

1. Acetone (Vo) 2. Naphthalene 3. Acenaphthene

1

2

3

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min

Figure 2. A sample chromatogram of the Reversed-Phase QCRM on an ACQUITY UPLC HSS C18, 2.1 x 50 mm, 1.7 μm column, held at 30 °C. The separation is isocratic using 65:35 Methanol: 20 mM phosphate buffered mobile phase at pH 7. The injection volume is 1.5 μL. The method uses a flow rate of 0.25 mL/min and a UV detection wavelength of 254 nm.

1. Uracil2. Propranolol3. Butylparaben4. Dipropylphthalate5. Naphthalene6. Acenaphthene7. Amitriptyline

1

2

3

4

5

6

7

0

0.00

0.02

0.04

0.06

0.08AU

0.10

0.12

0.14

0.16

2 4 6 8 10 12 14 16 18 20 22 24 min

NEUT RALS QC RM

The Neutrals QCRM is extremely versatile and appropriate for

a vast variety of column chemistries and dimensions, and system

hardware. It is adaptable to a wide variety of separation methods.

The injected quantity should be scaled for other column diameters.

To properly transfer the separation across column dimensions,

the ACQUITY UPLC Columns Calculator may be used. The

L/dp (length to particle size ratio) for both columns should be kept

comparable to maintain resolution.

The table below shows the three compounds that make up the

Neutrals QCRM. The first is a void volume marker, and the other two

are later eluting neutral compounds.

Compound

Acetone (V0)

Naphthalene

Acenaphthene

Table 1: Neutrals QCRM.

REV ERSED-PHASE QC RM

The Reversed-Phase QCRM contains 7 compounds including uracil,

butyl paraben, naphthalene, propranolol, dipropylphthalate,

acenaphthene, and amitriptyline at pH 7.

The compounds in the Reversed-Phase QCRM are listed in Table 2.

These compounds were chosen for their varying chemical properties

and retention on reversed-phase stationary phases. Under the

appropriate method conditions, the compounds can be resolved.

The Reversed-Phase QCRM is significantly more complex than

the Neutrals QCRM in that it contains compounds which could be

challenging to separate. It is designed to provide this complexity

and is appropriate when complex assays are routinely performed.

It can be used with a variety of column chemistries and dimensions,

and system hardware. On different column chemistries or dimensions,

methods may need to be modified or re-developed to obtain

sufficient resolution.

Compound Type

Amitriptyline Base

Acenaphthene Neutral

Naphthalene Neutral

Dipropyl phthalate Polar neutral

Butyl paraben Weak acid

Propranolol Base

Uracil Void marker

Table 2. The seven compounds contained in the Reversed-Phase QCRM.

3

Figure 3: TOF MS ES+ Spectrum of the LCMS QCRM Standard using methanol mobile phase with a SYNAPT® G2-S MS operated in positive ion mode in high resolution mode.

Acetaminophen

Caffeine

Sulfaguanidine

Sulfadimethoxine

Val -Tyr -Val

Verapamil

Terfenadine

Leucine-Enkephalin

Reserpine

LCMS QC RM

The LCMS QCRM is a 9 component mix used to provide a

comprehensive reference standard for use with LC/MS or MS

instrumentation with a wide variety of conditions and methods.

■■ The compounds in this mix give a mixture of responses in

ESi (+-) and APCi+.

■■ Covers a wide range of m/z.

■■ Optimized concentration to provide a more equal response by

component in ESI+ mode.

■■ Provides a separation in a range of chromatographic conditions

used to benchmark instrument performance.

Component Empirical Formula

Exact Mass(as [M+H]+)

Exact Mass(as [M+H]–)

Concentration for analysis

(µg/mL)

Acetaminophen C8H9NO2 152.0712 150.0555 10

Caffeine C8H10N4O2 195.0882 1.5

Sulfaguanidine C7H10N4O2S 215.0603 213.0446 5

Sulfadimethoxine C12H14N4O4S 311.0814 309.0658 1

Val-Tyr-Val C19H29N3O5 380.2185 378.2029 2.5

Verapamil C27H38N2O4 455.2910 0.2

Terfenadine C32H41NO2 472.3216 0.2

Leucine-Enkephalin C28H37N5O7 556.2771 554.2615 2.5

Reserpine C33H40N2O9 609.2812 0.6

Table 3. Individual Components in the LCMS QCRM Mix

HILIC QC RM

The HILIC QCRM was designed as a standard to help benchmark

this type of separation. It is a 4 component mix containing an

un-retained void marker, a polar neutral compound and two

polar basic compounds that provides a stable chromatographic

separation for HILIC columns and was specifically designed as a

batch test standard for ACQUITY UPLC® BEH HILIC columns.

Compound Name Property Concentration in Vial (mg/mL)

Acenaphthene V0 marker 0.0190

Thymine Polar neutral 0.0037

Adenine Polar basic 0.0037

Cytosine Polar basic 0.0077

Figure 4. Chromatographic Resolution using the HILIC QCRM.

AU

0.00

0.05

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

HILIC QCRMLot# W11061301

1

23

4

1. Acenaphthene 2. Thymine 3. Adenine 4. Cytosine

Table 4. HILIC QCRM Components

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

®2013. Waters, ACQUITY UPLC, ACQUITY, The Science of What’s Possible, SYNAPT, and UPLC are registered marks of Waters Corporation. Precise Formulation - Absolute Traceability - Only from Waters is a trademark of Waters Corporation.

©2013 Waters Corporation. Produced in the U.S.A.September 2013 720004434EN KP-PDF

WAT ERS QUALIT Y COMMITMENT

Waters understands that the quality of the standards and reagents you use directly correlates to the

quality of your results and final products. That is why we have made analytical standards and reagents our

business. Our seal symbolizes Waters commitment to you.

Precise Formulation

In the field of analytical measurement, the ability to repeatedly prepare reference materials identically

over long periods of time is critical for data comparability and defensibility.

Absolute Traceability

In order to be true, the properties of the measurement must be directly linked to a source reference material

through an unambiguous, unbroken and fully documented chain of comparisons.

Waters recommends that customers benchmark their chromatographic system with a QCRM prior to system usage

when it is in good working order. Run and compare the QCRM results to previous benchmarks before running

any critical assay, and after any hardware, column or mobile phase changes.

ORDERING INFORMATION

Description Part No.

Neutrals QC Reference Material 186006360

Reversed-Phase QC Reference Material 186006363

LCMS QC Reference Material 186006963

HILIC QC Reference Material 186007226

Preparative Chromatography Standard 186006703